Involvement of TGFBI-TAGLN axis in cancer stem cell property of head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a significant healthcare burden globally. Previous research using single-cell transcriptome analysis identified TGFBI as a crucial marker for the partial-epithelial-mesenchymal transition (partial-EMT) program. However, the precise role of TGFBI in HNSCC progression remains unclear. Therefore, our study aimed to clarify the impact of TGFBI on the malignant behavior of HNSCC cells. Through RNA-sequencing data from the TCGA database, we validated that increased TGFBI expression correlates with a higher occurrence of lymph node metastasis and unfavorable prognosis in HNSCC cases. Functional experiments demonstrated that TGFBI overexpression enhances the ability of sphere formation, indicating stem-cell-like properties. Conversely, TGFBI depletion reduces sphere formation and suppresses the expression of cancer stem cell (CSC) markers. RNA-sequencing analysis of TGFBI-overexpressing and control HNSCC cells revealed TAGLN as a downstream effector mediating TGFBI-induced sphere formation. Remarkably, TAGLN depletion abolished TGFBI-induced sphere formation, while its overexpression rescued the suppressed sphere formation caused by TGFBI depletion. Moreover, elevated TAGLN expression showed correlations with the expression of TGFBI and partial-EMT-related genes in HNSCC cases. In conclusion, our findings suggest that TGFBI may promote CSC properties through the upregulation of TAGLN. These novel insights shed light on the involvement of the TGFBI-TAGLN axis in HNSCC progression and hold implications for the development of targeted therapies.


Western Blot analysis
Western blotting was performed as described previously 11 .TGFBI-overexpressing Ho-1-U-1 and HSC3 cells were treated with 3 μM of monensin (Sigma, Burlington, USA) for 24 h.The intracellular protein expression of TGFBI is low because of the secretion from intracellular into extracellular.To quantify intracellular protein expression of TGFBI, we utilized monensin known as a protein transport inhibitor.Monensin blocks intracellular protein transport processes of any secreted proteins and induces the accumulation of these proteins in the Golgi complex.The increased accumulation of secreted proteins enhances the detectability of TGFBI with Western blot analysis utilizing total cell lysate.Cells were lysed using lysis buffer (50 mM pH 7.6 Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 0.5% Nonidet P-40, and 10% glycerol) with protease inhibitor cocktail (Nacalai tesque, Kyoto, Japan).After centrifugation, the supernatant was collected.Protein concentration was measured using Thermo Fischer BSA protein assay reagent (Thermo Scientific) by the absorption at 562 nm using a microplate reader (TECAN infinite 200Pro).Using 5-20% gradient polyacrylamide gel (ATTO Corporation, Tokyo, Japan), these proteins were electrophoresed followed by blotting onto a nitrocellulose membrane (GE Healthcare Life Science).By using a Western ECL Substrate (BIO-RAD), the signal was detected by an Amersham ImageQuant 800 (GE Healthcare Life Science).

Plasmids and transfection
For generating TGFBI-overexpressing cells, pcDNA3.1 + -C-(k)DYK plasmid encoding full-length human TGFBI (GenScript) was transfected into HNSCC cells by using Fugene HD (promega), according to the manufacturer's instructions.After transfections, we treated the cells with 500 µg/mL of G418, and then we picked up clones (clone and pool clone).

Sphere formation assay
HNSCC cells were seeded into 6-well Ultra low attachment surface plates (Corning, 5 × 10 4 cells/well) or 24-well Ultra low attachment surface plates (1 × 10 4 /well).After 4-7 days, the cells were cultured to allow the generation of spheroids.After generating spheroids, we counted the number of spheroids with a size of 100 µm or more, which is visible for naked eyes.

In vitro invasion assay
In vitro invasion assay was described previously 11 .Invasiveness was determined by using a 24-well cell culture insert with 8 µm pores (#3097, Becton Dickinson).The filter was coated with 50 µg Matrigel (Becton Dickinson) as a reconstituted basement membrane substance.The lower compartment contained 0.5 mL of culture media.Following trypsinization, 1.5 × 10 5 cells were plated on the upper compartment of the cell culture insert with 100 μL culture media.After 24 h incubation, the cells on the upper surface of the filter were removed by wiping with a cotton swab, and the cells on the lower surface of the filter were fixed with formalin and stained with hematoxylin (FUJIFILM Wako).The number of invaded cells (cells on the lower surface of the filter) was counted under a light microscope.
Bulk RNA-seq analysis mRNA was isolated from total RNA using the KAPA mRNA Capture kit (KAPA, cat.KK8440) and cDNA libraries were prepared using the MGIEasy RNA Directional Library Prep Set (MGI Tech, cat.1,000,006,385).Sequencing was performed using DNBSEQ-G400RS with a 150-bp paired-end configuration.The quality check and adaptor removal were performed using FastQC and fastp programs on the raw read sequences.The paired-end reads were mapped to the human reference genome (GRCh38 primary assembly genome) using the STAR software.After mapping, read counts and TPM values were generated using the RSEM software.Raw RNA-seq data (FASTQ files) reported in this study are available at the DDBJ Sequence Read Archive (DRR486141-DRR486143).

scRNA-seq analysis
The log-transformed gene expression values from single-cell RNA-seq data from HNSC (GSE103322) was analyzed using the Seurat (v4.3.0).As the quality control, genes expressed in less than 5 cells and cells with more than 10,000 genes were filtered.We performed dimensionality reduction using Uniform Manifold Approximation and Projection (UMAP) with the top 14 principal components.The resolution parameter was set to 0.4 for the FindClusters function.We determined the cell types of each cluster based on the marker genes identified using the FindAllMarkers function.

Data analysis
Clinical and gene expression information of 514 head and neck squamous cell carcinoma (TCGA-HNSC) samples were obtained from the cBioPortal website (https:// www.cbiop ortal.org/) for primary lymph node presentation assessment analysis, neoplasm histologic grade analysis, gene expression analysis, and survival analysis.The five-year overall survival of HNSC patients in the top 25% and bottom 25% expression groups for TGFBI, TAGLN, and TGFBI/TAGLN was analyzed using the survival and survminer R packages.The gene expressions (partial-EMT related genes, NANOG, POU5F1, SOX2, ALDH1, BCL11B, S100A4, VIM, TGFBI, and TAGLN) of HNSCC patients in the top 25% and bottom 25% expression groups for TGFBI, TAGLN, or TGFBI/TAGLN was analyzed on cBioPortal website.Also, the correlations of TGFBI expression with several gene expressions (FN1, LGALS3BP, MT1F, TAGLN, MME) in HNSCC patients were analyzed on cBioPortal website.The logtransformed TPM values of HNSC cell lines (DepMap 23Q2 files) were obtained from the depmap portal website (https:// depmap.org/ portal/).The scores of six gene programs (CellCycle, Epidif.1/2,Stress, Hypoxia, and pEMT) in each HNSC cell line were calculated using ssGSEA with the ConsensusTME R package.

Statistical analysis
For Kaplan-Meier overall survival, the log rank test was used for comparing two groups.For sphere formation assay, the Welch's t-test or ANOVA test was used for comparing groups.For in vitro invasion assay, chemoresistance assay, the Welch's t-test was used for comparing groups.For quantitative RT-PCR analysis, the t-test was used for comparing to control value.For correlation of gene expression using TCGA database, the Student's t-test was used for comparing two group.

Correlation of TGFBI expression with partial-EMT phenotype in HNSCC cell lines
To assess the correlation between TGFBI expression, lymph node metastasis, and prognosis in HNSCC cases, we analyzed processed RNA-seq data from the TCGA database.Notably, high TGFBI expression exhibited a higher incidence of lymph node metastasis compared with low TGFBI expression (Supplementary Fig. S1A).

Enhanced sphere formation by TGFBI in HNSCC cells
To elucidate the role of TGFBI, we generated TGFBI-overexpressing cells using Ho-1-U-1 and HSC3 cells with low TGFBI expression (Fig. 1A).We obtained both TGFBI-overexpressing clone and pool clone of Ho-1-U-1 and HSC3 cells.Compared to control cells, TGFBI-overexpressing cells exhibit a sheet-like cell mass with a smooth margin shape (Supplementary Fig. S3A).While TGFBI overexpression suppressed cell proliferation and invasion (Supplementary Fig. S3B,C), it significantly enhanced sphere formation ability (Fig. 1B).Sphereforming culture is well known for effectively enriching subpopulations with stem-cell properties.Indeed, TGFBIoverexpressing cells exhibited an increased number of colonies even when plated with a small number of cells (1000 cells/well and 500 cells/well) (Supplementary Fig. S4).To further demonstrate the phenotype induced by TGFBI overexpression, we utilized validated TGFBI siRNA in OSC20 and SAS cells with high TGFBI expression (Supplementary Figure S2A,B).Treatment with TGFBI siRNA effectively reduced TGFBI expression in both cell lines (Fig. 1C).Subsequently, we examined sphere formation in these cells and found that TGFBI depletion significantly decreased the number of colonies formed (Fig. 1D).
Thus, TGFBI was involved in sphere formation of HNSCC cells, indicating stem-cell-like properties.Indeed, SCC-4 cells with a partial-EMT phenotype showed higher expression of stem cell and CSC markers compared to other HNSCC cell lines (Supplementary Fig. S5A).Therefore, we investigated the expression of stemness markers, such as NANOG, OCT4, and SOX2 following the overexpression of TGFBI (Ho-1-U-1 and HSC3 cells).Although TGFBI overexpression tended to elevate the expression levels of stemness markers, we did not observe a significant difference (Supplementary Fig. S5B).Moreover, we examined CSC markers, ALDH1 and BCL11B in TGFBI-overexpressing cells (Ho-1-U-1 and HSC3) and TGFBI-depleted cells (OSC20 and SAS).TGFBI overexpression tended to elevate the expression levels of CSC markers and TGFBI depletion exhibited reduced expression of CSC markers (Supplementary Fig. S5C,D).

Involvement of TAGLN in TGFBI-promoted sphere formation
To pursuit unraveling the molecular mechanisms behind TGFBI-mediated sphere formation, we compared gene expression profiles of TGFBI-overexpressing clone and pool Ho-1-U-1 cells with control Ho-1-U-1 cells by RNA-sequencing analysis (Fig. 2A).TGFBI overexpression resulted in the upregulation of genes involved in negative regulation of cell population proliferation, angiogenesis, response to oxidative stress, and others, while downregulating genes involved in the regulation of GTPase activity, Wnt signaling pathway, organelle assembly Ras signaling, and more (Supplementary Fig. S6).Among the upregulated genes in TGFBI-overexpressing cells, we checked the genes with high expression of TGFBI in HNSCC cases from the TCGA database (Fig. 2B,C).FN1 (Fibronectin 1), LGALS3BP (galectin 3 binding protein), MT1F (metallothionein 1F), TAGLN (transgelin), and MME (membrane metalloendopeptidase) were identified as a gene with a strong correlation with TGFBI (Fig. 2C,D).Notably, only TAGLN was significantly downregulated by TGFBI depletion in HNSCC cells (Fig. 2E).Furthermore, we confirmed that TAGLN expression was upregulated by TGFBI-overexpressing cells (Fig. 2F).TAGLN depletion decreased TGFBI expression, that it may be caused by positive feedback (Supplementary Figure S7A).
Subsequently, we further investigated the involvement of TAGLN in TGFBI-mediated sphere formation.TAGLN shRNA reduced the expression levels of TAGLN mRNA and protein (Fig. 3A,B).As expected, TAGLN depletion suppressed sphere formation (Fig. 3C,D).However, TAGLN depletion did not decrease CSC markers, ALDH1 and BCL11B (Supplementary Figure S7B).Moreover, we examined the impact of TAGLN depletion on sphere formation in TGFBI-overexpressing Ho-1-U-1 cells (Fig. 3A,B).The depletion of TAGLN suppressed sphere formation, as did the sphere formation induced by TGFBI overexpression (Fig. 3C,D).Additionally, we investigated the effect of TAGLN overexpression in TGFBI-depleted SAS and OSC20 cells (Fig. 3E).Interestingly, TAGLN overexpression not only promoted sphere formation on its own but also rescued the sphere formation suppressed by TGFBI depletion (Fig. 3F,G).Given that TGFBI contains an RGD sequence that binds to integrin αvβ3 and αvβ5, with a higher affinity for the αvβ5 complex 12,13 , we explored its potential role in integrin signaling.TGFBI is known as the primary intracellular downstream signaling mediators of integrins and promotes αvβ5 integrin signaling to FAK (focal adhesion kinase), thereby contributing to cancer progression through its integrin-binding RGD motif in osteosarcoma, colon, and pancreatic cancer models 12,14,15 .To investigate this mechanism, we treated TGFBI-overexpressing Ho-1-U-1 cells with the FAK inhibitor 14, which directly inhibits FAK Y397 autophosphorylation.FAK inhibitor downregulated TAGLN expression in a dose-dependent manner (Fig. 3H,I), suggesting that TAGLN expression may be induced through TGFBI-mediated integrin signaling.

Correlation of TAGLN expression with clinical parameters and TGFBI in HNSCC
To investigate the correlation of TAGLN expression with lymph node metastasis and prognosis in HNSCC cases, we analyzed processed RNA-seq data obtained from the TCGA database.High expression of TAGLN was associated with advanced grading, lymph node metastasis, and poor prognosis, compared to low expression of TAGLN (Supplementary Fig. S8A and B).Although TAGLN expression was dominantly observed in cancer stromal fibroblasts, a certain population of cancer cells also expressed TAGLN (Supplementary Fig. S8C).This result is consistent with the notion that cancer stem cells are a small population within the cancer cells.Additionally, we examined the relationship between TAGLN and TGFBI expression in HNSCC cases, revealing that high expression of TAGLN was correlated with elevated TGFBI expression, and vice versa (Fig. 4A).Moreover, we examined the correlation between TAGLN and partial-EMT-related genes identified in a previous study 8 .Interestingly, HNSCC cases with high TAGLN expression exhibited increased expression of partial-EMTrelated genes (Fig. 4B).Furthermore, we explored the correlation of TGFBI/TAGLN expression with stem cell markers (NANOG, POU5F1, SOX2, ALDH1, and BCL11B) and EMT markers (S100A4 and VIM) in HNSCC cells.There was no significant elevation in stem cell markers in HNSCC cases with high expression of TGFBI and TAGLN compared to those with low expression of these genes (Supplementary Figure S9).However, EMT markers exhibited higher levels in HNSCC cases with high TGFBI and TAGLN expression (Supplementary Figure S9).We conducted Kaplan-Meier overall survival analysis in HNSCC patients with "low" and "high" TGFBI/TAGLN expression.Interestingly, HNSCC patients with high TGFBI/TAGLN expression significantly exhibited an unfavorable prognosis compared to those with low expression of these genes (Fig. 4C).

Discussion
TGFBI, belonging to the FAS1 family, shares a striking 48% similarity with periostin, a secreted protein renowned for its influential impact 16 .Like TGFBI, periostin contains the EMI and four FAS1 domains, but does not contain an RGD sequence.Our previous study demonstrated that periostin has the ability to promote invasion, angiogenesis, and metastasis in HNSCC 11,17 .TGFBI does not enhance migration and invasion but remarkably promotes sphere formation-an intriguing distinction in its function within HNSCC cells despite their similar protein structures.
Although previous reports have highlighted the dual nature of TGFBI, acting both as a tumor suppressor and a promoter, accumulating evidence underscores its significant effects in driving tumor progression 10 .Single cell transcriptome analysis identifies TGFBI as a marker of partial-EMT 8 .Moreover, bioinformatics analysis pinpoints its identification as a hub gene associated with lymph node metastasis in HNSCC 18 .Also, we previously have shown a significant correlation between TGFBI expression and poor survival among HNSCC patients 19 .Thus, TGFBI acts as a promoter of tumor progression in HNSCC.TGFBI's impact extends beyond mere correlations, encompassing critical aspects such as chemotaxis, migratory potential, proliferation, apoptosis, metastatic niche promotion, cancer cell adhesion, and aberrant angiogenesis in other types of cancer 10,14,15,[20][21][22][23][24][25][26][27] .Mechanistically, TGFBI propels cancer progression by activating αvβ5 integrin signaling, ultimately engaging Src, FAK, PI3K, and AKT through its RGD motif, as observed in models of osteosarcoma, colon, and pancreatic cancer 12,14,15 .Indeed, TGFBI is identified as a necessary gene for tumorsphere formation in stem-like breast cancer cells expressing the integrin αvβ3 28 .Although previous study shows that TGFBI-positive HNSCC cells sorted by flow cytometry exhibit increased invasiveness and decreased proliferation 8 , here we demonstrate that TGFBI remarkably enhances the ability of HNSCC cells to form spheres, a hallmark of CSC properties.
In our pursuit of unraveling the molecular mechanisms behind TGFBI-mediated sphere formation, we conducted RNA sequencing analysis, which revealed TAGLN as a gene upregulated in TGFBI-overexpressing cells and highly correlated with TGFBI expression in HNSCC cases from the TCGA database.TAGLN plays pivotal roles in podosome formation, myocyte migration, and vascular and visceral smooth muscle cell differentiation 29 .Although TAGLN is traditionally considered a tumor suppressor in various cancers, our study uncovers elevated TAGLN expression closely associated with adverse clinical parameters, including poor survival, advanced grading, and lymph node metastasis in HNSCC patients.Furthermore, TAGLN expression exhibits a strong correlation with the expression of TGFBI and partial-EMT-related genes, suggesting a crucial role for TAGLN in the aggressiveness and metastatic potential of HNSCC mediated by TGFBI.While the intricate mechanisms governing TAGLN regulation by TGFBI remain elusive, our study hints at the involvement of integrin signaling and its downstream effector, FAK.Notably, the inhibition of FAK downregulates TAGLN expression, suggesting that TGFBI-mediated integrin signaling, facilitated by its RGD motif, may induce TAGLN expression in HNSCC cells.Intriguingly, TGFBI-positive cells exhibiting partial-EMT traits localize at the leading edge of HNSCC tissue 8 , implying potential interactions between TGFBI and the tumor microenvironment, particularly ligand-receptor signaling.Furthermore, TGFBI exhibits enhanced sphere formation with upregulation of CSC markers.However, TAGLN depletion did not contribute to the expression of CSC markers.The correlation between TGFBI-TAGLN axis and stemness is still unknown.The detailed mechanism on the involvement of TGFBI in regulating tumor microenvironment and stemness requires further study.
In conclusion, our study uncovers a TGFBI-TAGLN axis via integrin signaling, underscoring their potential interplay in driving HNSCC.The association of TGFBI and TAGLN with adverse clinical parameters further emphasizes its significance in HNSCC aggressiveness.Our investigations have illuminated the captivating involvement of TGFBI-TAGLN axis in HNSCC, positioning them as promising targets for future therapies. https://doi.org/10.1038/s41598-024-57478-0www.nature.com/scientificreports/

Figure 1 .
Figure 1.Sphere formation by TGFBI in HNSCC cells.(A) Immunoblotting confirming ectopic TGFBI expression in TGFBI-overexpressing Ho-1-U-1 and HSC3 cells (clone and pool cells for each cell line).β-actin served as the loading control.(B) Assessment of sphere formation using Ultra low attachment surface plates.Control, clone, and pool TGFBI-overexpressing Ho-1-U-1 and HSC3 cells were employed.5 × 10 4 cells were seeded into 6-well Ultra low attachment surface plates.Images of spheroid formation are shown after 4 days (Ho-1-U-1 cells) and 6 days (HSC3 cells) in upper panel.Lower panel shows the number of spheroids with a size of 100 µm or more after 4 days (Ho-1-U-1 cells) and 6 days (HSC3 cells).Data represent the mean ± SD in each group (n = 4).*P-value < 0.05.(C) Quantitative RT-PCR analysis of TGFBI expression in OSC20 and SAS cells transfected with TGFBI siRNA.*P-value < 0.05.(D) Assessment of sphere formation using Ultra low attachment surface plates.Control and TGFBI-knockdown OSC20 and SAS cells were used.5 × 10 4 cells were seeded into 6-well Ultra low attachment surface plates.Images of spheroid formation are shown after 6 days in upper panel.Lower panel shows that the number of spheroids with a size of 100 µm or more after 6 days.Data represent the mean ± SD of triplicates in each group.*P-value < 0.05.

Figure 3 .
Figure 3. Involvement of TAGLN in TGFBI-promoted sphere formation.(A) Quantitative RT-PCR analysis of TAGLN expression in TAGLN shRNA-transfected TGFBI-overexpressing Ho-1-U-1 cells and control Ho-1-U-1 cells.Data represent the mean ± SD of triplicates in each group.*P-value < 0.05.(B) Immunoblotting confirming the expression of TAGLN in TGFBI-overexpressing Ho-1-U-1 cells transfected with TAGLN shRNA.β-actin served as the loading control.(C) Assessment of sphere formation using Ultra low attachment surface plates. 1 × 10 4 cells were seeded into 24-well Ultra low attachment surface plates.Images of spheroid formation are shown after 4 days.(D) The number of spheroids with a size of 100 µm or more was counted after 4 days.Data represent the mean ± SD of triplicates in each group.*P-value < 0.05.(E) Immunoblotting confirming the expression of HA-tagged TAGLN and TGFBI in TAGLN-overexpressing SAS and OSC20 cells transfected with TGFBI siRNA.β-actin served as the loading control.(F) Assessment of sphere formation using Ultra low attachment surface plates. 1 × 10 4 cells were seeded into 24-well Ultra low attachment surface plates.Images of spheroid formation are shown after 6 days.(G) The number of spheroids with a size of 100 µm or more was counted after 6 days.Data represent the mean ± SD in each group (n = 4).*P-value < 0.05.(H) TGFBI-induced TAGLN expression mediated by FAK.TGFBI-overexpressing Ho-1-U-1 cells showed a decreased expression of TAGLN, when treated by FAK inhibitor 14 at indicated concentration.After 3 h treatment, cells were collected and TAGLN expression was determined by quantitative RT-PCR.Data represent the mean ± SD of triplicates in each group.*P-value < 0.05.(I) The expression of TAGLN was examined by immunoblotting after treatment FAK inhibitor 14 at indicated concentration.β-actin served as the loading control.

Figure 4 .
Figure 4. Correlation of TAGLN expression with the expression of TGFBI and partial-EMT -related genes in HNSCC.(A) Correlation between TAGLN expression and TGFBI expression in HNSCC patients.*P-value < 0.05.(B) Correlation between TAGLN expression and the expression of partial-EMT-related genes in HNSCC patients.*P-value < 0.05.(C) Kaplan-Meier overall survival analysis in HNSCC patients with "low" and "high" TGFBI/TAGLN expression.HNSCC cases were divided into two groups based on both TGFBI and TAGLN expression levels: "low" (n = 50, bottom 25% patient group) and "high" (n = 78, top 25% patient group).